Project Summary: The arenavirus Lassa (LASV) infects several hundred thousand individuals yearly in West Africa resulting in a high number of Lassa fever (LF) cases that are associated with high morbidity and mortality. In addition, LASV is a credible biodefense threat. There are not FDA-licensed vaccines and current anti-arenaviral therapy is limited to the off-label use of ribavirin that is only partially effective. The significance of LASV in human health and biodefense readiness, together with the limited existing armamentarium to combat them, underscore the unmet need for novel anti-LASV therapeutics. We hypothesize that selective targeting of host cell factors required for virus RNA replication and gene transcription, processes mediated by the virus ribonucleoprotein (vRNP), but dispensable for normal host cell metabolism and survival, represents a novel strategy to combat human pathogenic arenaviruses. This approach would minimize the common problem in antiviral therapy posed by the emergence of drug resistant variants. To implement this strategy, we propose to conduct a siRNA genome-wide screen to identify host cell genes required for the activity of LASV vRNP. For this we have generated a virus-free cell line containing a functional LASV vRNP (LASV/vRNP) that directs expression of the ZsGreen and Gaussia luciferase (Gluc) reporter genes (RG) from a virus-like RNA, aka minigenome (MG), and shown that RG expression (RGE) accurately reflect vRNP activity. Our specific aims (SA) are: SA 1. Conduct siRNA-based genome-wide screen to identify host cell factors that contribute to the activity of LASV vRNP. We have used our LASV/vRNP cells to establish conditions compatible with genetic HTS to identify modifiers of LASV vRNP activity. We propose to screen our LASV/vRNP cells against Dharmacon?s On-Target-Plus human genome library to identify host cell genes that inhibit LASV vRNP activity as reflected by reduction of Gluc and ZsGreen readings. SA 2. Hit validation. We will determine levels of LASV MG-directed expression levels of a different reporter gene, CAT, in cells where hit candidates have been subjected to RNAi-mediated knock down, complete loss- of-function via CRISPR-Cas9, and pharmacological interference. To assess hit specificity we will perform MG assays with other RNA viruses. Further hit validation will be done using cell types that are biologically relevant in the context of LASV including human epithelial and endothelia cells, hepatocytes and monocytes. Knowledge derived from these studies will provide: 1) the foundation for designing antiviral strategies aimed at targeting host cell factors required for essential viral RNA biosynthetic processes, and 2) novel insights about arenavirus-host interactions that will contribute to a better understanding of arenavirus pathogenesis.